Gene activation and silencing are critical biological processes. Histone proteins, the basic building blocks of chromatin, influence gene expression through intimate contact with DNA and with the transcription machinery. Chromatin assembly is a highly regulated, essential process that involves the coordination of the DNA replication machinery with assembly factors and histones, and is critical for the formation of silent chromatin. N-terminal histone H4 mutants (h4u) disrupt the formation of silent chromatin in yeast, yet are recessive when a wild-type gene is also present. Preliminary data suggests that h4u is excluded from chromatin and that Chromatin Assembly Factor-I contributes to this exclusion. To test the hypothesis that cells actively prevent h4u incorporation into chromatin, ratios of wild type to h4u will be measured in whole cells, in chromatin and in silent regions of the genome. The relative contributions of nucleosome assembly factors towards h4u exclusion will also be examined. Finally, the molecular mechanisms by which nucleosome assembly factors exclude h4u from chromatin will be investigated by determining if h4u is sequestered by assembly factors and/or if h4u is incorporated into specific chromosomal domains. These studies will improve understanding of the fundamental cellular mechanisms of chromatin assembly and silent chromatin formation.